Isolated human enzyme proteins, nucleic acid molecules encoding human enzyme proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of enzyme proteins that arerelated to the NADPH oxidase subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins that effect protein phosphorylation and nucleicacid molecules encoding such peptide and protein molecules, all of whichare useful in the development of human therapeutics and diagnosticcompositions and methods.

BACKGROUND OF THE INVENTION

[0002] Many human enzymes serve as targets for the action ofpharmaceutically active compounds. Several classes of human enzymes thatserve as such targets include helicase, steroid esterase and sulfatase,convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase,glutanase, decarboxylase, isomerase and reductase. It is thereforeimportant in developing new pharmaceutical compounds to identify targetenzyme proteins that can be put into high-throughput screening formats.The present invention advances the state of the art by providing novelhuman drug target enzymes related to the NADPH oxidase subfamily.

[0003] Neutrophil NADPH oxidase is a multicomponent enzyme that isactivated to generate superoxide anion and is defective in the cells ofpatients with chronic granulomatous disease. It requires both membraneand cytosolic components, the latter including 47- and 67-kDa proteinsrecognized by the polyclonal antiserum B-1. Immunoscreening of aninduced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizingcDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa wererecognized by B-1. Antiserum against a fusion protein recognized a47-kDa protein in normal neutrophils but not in those from patients withautosomal chronic granulomatous disease who lack the 47-kDa cytosolicoxidase component.

[0004] The phagocyte NADPH oxidase is a complex enzyme system that playsan important role in host defense. After stimulation with opsonizedmicroorganisms or other activating agents, the oxygen consumption ofthese cells increases dramatically (respiratory burst) and they releasea large amount of superoxide. Superoxide is then converted to morepotent reactive oxygen species such as hydrogen peroxide, hydroxylradical, and hypohalous acids, which are used by phagocytes to controlmicrobial infections. The importance of this defense mechanism is madeevident by a rare inherited syndrome, chronic granulomatous disease(CGD) in which phagocytes fail to generate superoxide, rendering thepatients highly susceptible to life-threatening microbial infections.

[0005] The present invention has a substantial similarity to p47phox.The cDNA of this enzyme was originally isolated for the 47-kilodalton(kDa) subunit of the NADPH oxidase system, whose absence is responsiblefor the most common form of autosomally inherited chronic granulomatousdisease (CGD). It encodes a 44.7-kDa polypeptide, which contains two srchomology (SH3) domains and several possible sites for phosphorylation byprotein kinase C. An antiserum raised to the predicted C terminus of theprotein detects a polypeptide with an apparent molecular mass of 47 kDain normal neutrophil granulocytes but not in those from patients withautosomal CGD. The antibody has been used to show that the proteinassociates with the vacuolar membrane and is phosphorylated in responseto phorbol ester treatment. Analysis of a number of tissue types andcell lines shows that expression of the gene is confined to phagocyticcells and B lymphocytes. It is suggested that patients with CGD may alsohave a defect in lymphocyte function. p47 protein and mRNA levelsincrease during retinoic acid-induced neutrophil differentiation of HL60cells. The gene is a primary target for regulation by retinoic acid. Fora review related to p47 phox, see Rodaway et al., Mol Cell Biol 1990October;10(10):5388-96; Volpp et al., Proc Natl Acad Sci U S A 1989September;86(18):7195-9 Erratum in: Proc Natl Acad Sci U S A 1989December;86(23):9563.

[0006] Enzyme proteins, particularly members of the NADPH oxidasesubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of enzyme proteins. The present invention advances the stateof the art by providing previously unidentified human enzyme proteins,and the polynucleotides encoding them, that have homology to members ofthe NADPH oxidase subfamily. These novel compositions are useful in thediagnosis, prevention and treatment of biological processes associatedwith human diseases.

SUMMARY OF THE INVENTION

[0007] The present invention is based in part on the identification ofamino acid sequences of human enzyme peptides and proteins that arerelated to the NADPH oxidase subfamily, as well as allelic variants andother mammalian orthologs thereof. These unique peptide sequences, andnucleic acid sequences that encode these peptides, can be used as modelsfor the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate enzyme activity incells and tissues that express the enzyme. Experimental data as providedin FIG. 1 indicates expression in humans in the placenta, B cells fromBurkitt lymphoma, primary B-cells from tonsils and leukocyte.

DESCRIPTION OF THE FIGURE SHEETS

[0008]FIG. 1 provides the nucleotide sequence of a cDNA molecule ortranscript sequence that encodes the enzyme protein of the presentinvention. (SEQ ID NO:1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. 1 indicates expression in humansin the placenta, B cells from Burkitt lymphoma, primary B-cells fromtonsils and leukocyte.

[0009]FIG. 2 provides the predicted amino acid sequence of the enzyme ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0010]FIG. 3 provides genomic sequences that span the gene encoding theenzyme protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs, including insertion/deletionvariants (“indels”), were identified at 41 different nucleotidepositions.

DETAILED DESCRIPTION OF THE INVENTION

[0011] General Description

[0012] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a enzyme protein or part of aenzyme protein and are related to the NADPH oxidase subfamily. Utilizingthese sequences, additional genomic sequences were assembled andtranscript and/or cDNA sequences were isolated and characterized. Basedon this analysis, the present invention provides amino acid sequences ofhuman enzyme peptides and proteins that are related to the NADPH oxidasesubfamily, nucleic acid sequences in the form of transcript sequences,cDNA sequences and/or genomic sequences that encode these enzymepeptides and proteins, nucleic acid variation (allelic information),tissue distribution of expression, and information about the closest artknown protein/peptide/domain that has structural or sequence homology tothe enzyme of the present invention.

[0013] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known enzyme proteins of theNADPH oxidase subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin the placenta, B cells from Burkitt lymphoma, primary B-cells fromtonsils and leukocyte. The art has clearly established the commercialimportance of members of this family of proteins and proteins that haveexpression patterns similar to that of the present gene. Some of themore specific features of the peptides of the present invention, and theuses thereof, are described herein, particularly in the Background ofthe Invention and in the annotation provided in the Figures, and/or areknown within the art for each of the known NADPH oxidase family orsubfamily of enzyme proteins.

[0014] Specific Embodiments

[0015] Peptide Molecules

[0016] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theenzyme family of proteins and are related to the NADPH oxidase subfamily(protein sequences are provided in FIG. 2, transcript/cDNA sequences areprovided in FIG. 1 and genomic sequences are provided in FIG. 3). Thepeptide sequences provided in FIG. 2, as well as the obvious variantsdescribed herein, particularly allelic variants as identified herein andusing the information in FIG. 3, will be referred herein as the enzymepeptides of the present invention, enzyme peptides, or peptides/proteinsof the present invention.

[0017] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the enzyme peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0018] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0019] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0020] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theenzyme peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0021] The isolated enzyme peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the placenta, B cells from Burkitt lymphoma, primary B-cellsfrom tonsils and leukocyte. For example, a nucleic acid moleculeencoding the enzyme peptide is cloned into an expression vector, theexpression vector introduced into a host cell and the protein expressedin the host cell. The protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Many of these techniques are described in detail below.

[0022] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0023] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0024] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the enzyme peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0025] The enzyme peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a enzyme peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the enzyme peptide. “Operatively linked”indicates that the enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the enzyme peptide.

[0026] In some uses, the fusion protein does not affect the activity ofthe enzyme peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant enzyme peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0027] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the enzyme peptide.

[0028] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0029] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the enzyme peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0030] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0031] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0032] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0033] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the enzyme peptides of the present invention as well as beingencoded by the same genetic locus as the enzyme peptide provided herein.As indicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 19 by ePCR.

[0034] Allelic variants of a enzyme peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the enzyme peptide as well asbeing encoded by the same genetic locus as the enzyme peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 19 by ePCR. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a enzyme peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0035]FIG. 3 provides information on SNPs that have been identified in agene encoding the enzyme protein of the present invention. 41 SNPvariants were found, including 7 indels (indicated by a “−”) and 4 SNPsin exons, of which 2 of these cause changes in the amino acid sequence(i.e., nonsynonymous SNPs). The changes in the amino acid sequence thatthese SNPs cause is indicated in FIG. 3 and can readily be determinedusing the universal genetic code and the protein sequence provided inFIG. 2 as a reference SNPs, identified at different nucleotide positionsin introns and regions 5′ and 3′ of the ORF, may affectcontrol/regulatory elements.

[0036] Paralogs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a enzymepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0037] Orthologs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0038] Non-naturally occurring variants of the enzyme peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the enzyme peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a enzyme peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0039] Variant enzyme peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0040] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0041] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as enzyme activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0042] The present invention further provides fragments of the enzymepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0043] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a enzyme peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the enzyme peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe enzyme peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0044] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inenzyme peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0045] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0046] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0047] Accordingly, the enzyme peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature enzyme peptide is fused withanother compound, such as a compound to increase the half-life of theenzyme peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature enzyme peptide or a pro-protein sequence.

[0048] Protein/Peptide Uses

[0049] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a enzyme-effectorprotein interaction or enzyme-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0050] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0051] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, enzymes isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the enzyme. Experimental data as providedin FIG. 1 indicates that the enzymes of the present invention areexpressed in humans in the placenta, B cells from Burkitt lymphoma,primary B-cells from tonsils detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression in andleukocyte. A large percentage of pharmaceutical agents are beingdeveloped that modulate the activity of enzyme proteins, particularlymembers of the NADPH oxidase subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in the placenta, B cells fromBurkitt lymphoma, primary B-cells from tonsils and leukocyte. Such usescan readily be determined using the information provided herein, thatwhich is known in the art, and routine experimentation.

[0052] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to enzymes that are related tomembers of the NADPH oxidase subfamily. Such assays involve any of theknown enzyme functions or activities or properties useful for diagnosisand treatment of enzyme-related conditions that are specific for thesubfamily of enzymes that the one of the present invention belongs to,particularly in cells and tissues that express the enzyme. Experimentaldata as provided in FIG. 1 indicates that the enzymes of the presentinvention are expressed in humans in the placenta, B cells from Burkittlymphoma, primary B-cells from tonsils detected by a virtual northernblot. In addition, PCR-based tissue screening panels indicate expressionin and leukocyte.

[0053] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the enzyme, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, B cells from Burkittlymphoma, primary B-cells from tonsils and leukocyte. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe enzyme protein.

[0054] The polypeptides can be used to identify compounds that modulateenzyme activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the enzyme.Both the enzymes of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the enzyme. These compounds can befurther screened against a functional enzyme to determine the effect ofthe compound on the enzyme activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the enzyme to a desired degree.

[0055] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the enzyme protein and a molecule that normally interacts withthe enzyme protein, e.g. a substrate or a component of the signalpathway that the enzyme protein normally interacts (for example, anotherenzyme). Such assays typically include the steps of combining the enzymeprotein with a candidate compound under conditions that allow the enzymeprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the enzymeprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0056] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0057] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantenzymes or appropriate fragments containing mutations that affect enzymefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0058] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) enzyme activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate enzyme activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the enzyme proteindependent signal cascade can be assayed.

[0059] Any of the biological or biochemical finctions mediated by theenzyme can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the enzyme can be assayed.Experimental data as provided in FIG. 1 indicates that the enzymes ofthe present invention are expressed in humans in the placenta, B cellsfrom Burkitt lymphoma, primary B-cells from tonsils detected by avirtual northern blot. In addition, PCR-based tissue screening panelsindicate expression in and leukocyte.

[0060] Binding and/or activating compounds can also be screened by usingchimeric enzyme proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native enzyme. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the enzyme is derived.

[0061] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the enzyme (e.g. binding partners and/or ligands).Thus, a compound is exposed to a enzyme polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble enzyme polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble enzyme polypeptide, itdecreases the amount of complex formed or activity from the enzymetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the enzyme.Thus, the soluble polypeptide that competes with the target enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

[0062] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the enzyme protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0063] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of enzyme-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a enzyme-binding protein and a candidate compound are incubated inthe enzyme protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theenzyme protein target molecule, or which are reactive with enzymeprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0064] Agents that modulate one of the enzymes of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0065] Modulators of enzyme protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the enzyme pathway, by treating cells or tissuesthat express the enzyme. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, B cells from Burkittlymphoma, primary B-cells from tonsils and leukocyte. These methods oftreatment include the steps of administering a modulator of enzymeactivity in a pharmaceutical composition to a subject in need of suchtreatment, the modulator being identified as described herein.

[0066] In yet another aspect of the invention, the enzyme proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the enzyme and are involved in enzyme activity.Such enzyme-binding proteins are also likely to be involved in thepropagation of signals by the enzyme proteins or enzyme targets as, forexample, downstream elements of a enzyme-mediated signaling pathway.Alternatively, such enzyme-binding proteins are likely to be enzymeinhibitors.

[0067] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a enzyme proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aenzyme-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the enzyme protein.

[0068] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a enzyme-modulating agent, an antisense enzymenucleic acid molecule, a enzyme-specific antibody, or a enzyme-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0069] The enzyme proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, B cells from Burkittlymphoma, primary B-cells from tonsils and leukocyte. The methodinvolves contacting a biological sample with a compound capable ofinteracting with the enzyme protein such that the interaction can bedetected. Such an assay can be provided in a single detection format ora multi-detection format such as an antibody chip array.

[0070] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0071] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered enzyme activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0072] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0073] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the enzyme protein in which one ormore of the enzyme functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and enzyme activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0074] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the placenta, B cells from Burkitt lymphoma,primary B-cells from tonsils and leukocyte. Accordingly, methods fortreatment include the use of the enzyme protein or fragments.

[0075] Antibodies

[0076] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0077] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0078] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0079] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0080] Antibodies are preferably prepared from regions or discretefragments of the enzyme proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or enzyme/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0081] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0082] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylanine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0083] Antibody Uses

[0084] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in theplacenta, B cells from Burkitt lymphoma, primary B-cells from tonsilsdetected by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in and leukocyte. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0085] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in the placenta, B cells from Burkittlymphoma, primary B-cells from tonsils and leukocyte. If a disorder ischaracterized by a specific mutation in the protein, antibodies specificfor this mutant protein can be used to assay for the presence of thespecific mutant protein.

[0086] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the placenta, B cells from Burkitt lymphoma, primary B-cells fromtonsils and leukocyte. The diagnostic uses can be applied, not only ingenetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0087] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0088] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in theplacenta, B cells from Burkitt lymphoma, primary B-cells from tonsilsand leukocyte. Thus, where a specific protein has been correlated withexpression in a specific tissue, antibodies that are specific for thisprotein can be used to identify a tissue type.

[0089] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the enzyme peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0090] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0091] Nucleic Acid Molecules

[0092] The present invention further provides isolated nucleic acidmolecules that encode a enzyme peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the enzyme peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0093] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0094] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0095] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0096] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0097] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0098] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0099] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0100] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0101] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the enzyme peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0102] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0103] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the enzyme proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0104] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0105] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0106] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0107] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 19 by ePCR.

[0108]FIG. 3 provides information on SNPs that have been identified in agene encoding the enzyme protein of the present invention. 41 SNPvariants were found, including 7 indels (indicated by a “−”) and 4 SNPsin exons, of which 2 of these cause changes in the amino acid sequence(i.e., nonsynonymous SNPs). The changes in the amino acid sequence thatthese SNPs cause is indicated in FIG. 3 and can readily be determinedusing the universal genetic code and the protein sequence provided inFIG. 2 as a reference SNPs, identified at different nucleotide positionsin introns and regions 5′ and 3′ of the ORF, may affectcontrol/regulatory elements.

[0109] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, New York (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2× SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0110] Nucleic Acid Molecule Uses

[0111] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs, including insertion/deletion variants(“indels”), were identified at 41 different nucleotide positions.

[0112] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0113] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0114] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0115] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0116] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome19 by ePCR.

[0117] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0118] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0119] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0120] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0121] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0122] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the enzymes of the present invention are expressed inhumans in the placenta, B cells from Burkitt lymphoma, primary B-cellsfrom tonsils detected by a virtual northern blot. In addition, PCR-basedtissue screening panels indicate expression in and leukocyte.Accordingly, the probes can be used to detect the presence of, or todetermine levels of, a specific nucleic acid molecule in cells, tissues,and in organisms. The nucleic acid whose level is determined can be DNAor RNA. Accordingly, probes corresponding to the peptides describedherein can be used to assess expression and/or gene copy number in agiven cell, tissue, or organism. These uses are relevant for diagnosisof disorders involving an increase or decrease in enzyme proteinexpression relative to normal results.

[0123] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0124] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a enzyme protein, such as bymeasuring a level of a enzyme-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a enzymegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans in theplacenta, B cells from Burkitt lymphoma, primary B-cells from tonsilsdetected by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in and leukocyte.

[0125] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate enzyme nucleic acid expression.

[0126] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the enzyme gene, particularly biological and pathologicalprocesses that are mediated by the enzyme in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in the placenta, B cells from Burkitt lymphoma, primaryB-cells from tonsils and leukocyte. The method typically includesassaying the ability of the compound to modulate the expression of theenzyme nucleic acid and thus identifying a compound that can be used totreat a disorder characterized by undesired enzyme nucleic acidexpression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing the enzymenucleic acid or recombinant cells genetically engineered to expressspecific nucleic acid sequences.

[0127] The assay for enzyme nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the enzyme proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0128] Thus, modulators of enzyme gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of enzyme mRNA inthe presence of the candidate compound is compared to the level ofexpression of enzyme mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0129] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate enzyme nucleic acid expressionin cells and tissues that express the enzyme. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the placenta, B cells from Burkitt lymphoma,primary B-cells from tonsils detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression in andleukocyte. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

[0130] Alternatively, a modulator for enzyme nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits theenzyme nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in the placenta, B cells from Burkitt lymphoma, primary B-cellsfrom tonsils and leukocyte.

[0131] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe enzyme gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0132] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in enzyme nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in enzyme genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the enzyme gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a enzyme protein.

[0133] Individuals carrying mutations in the enzyme gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been identified in a gene encoding theenzyme protein of the present invention. 41 SNP variants were found,including 7 indels (indicated by a “−”) and 4 SNPs in exons, of which 2of these cause changes in the amino acid sequence (i.e., nonsynonymousSNPs). The changes in the amino acid sequence that these SNPs cause isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a referenceSNPs, identified at different nucleotide positions in introns andregions 5′ and 3′ of the ORF, may affect control/regulatory elements. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 19 by ePCR. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0134] Alternatively, mutations in a enzyme gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0135] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0136] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant enzyme gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0137] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0138] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the enzyme gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been identified in a gene encoding theenzyme protein of the present invention. 41 SNP variants were found,including 7 indels (indicated by a “−”) and 4 SNPs in exons, of which 2of these cause changes in the amino acid sequence (i.e., nonsynonymousSNPs). The changes in the amino acid sequence that these SNPs cause isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a referenceSNPs, identified at different nucleotide positions in introns andregions 5′ and 3′ of the ORF, may affect control/regulatory elements.

[0139] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0140] The nucleic acid molecules are thus useful as antisenseconstructs to control enzyme gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of enzyme protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into enzyme protein.

[0141] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of enzyme nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired enzyme nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the enzyme protein, such as substratebinding.

[0142] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in enzyme geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired enzymeprotein to treat the individual.

[0143] The invention also encompasses kits for detecting the presence ofa enzyme nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in the placenta, B cells from Burkitt lymphoma,primary B-cells from tonsils detected by a virtual northern blot. Inaddition, PCR-based tissue screening panels indicate expression in andleukocyte. For example, the kit can comprise reagents such as a labeledor labelable nucleic acid or agent capable of detecting enzyme nucleicacid in a biological sample; means for determining the amount of enzymenucleic acid in the sample; and means for comparing the amount of enzymenucleic acid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect enzyme protein mRNA or DNA.

[0144] Nucleic Acid Arrays

[0145] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0146] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0147] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0148] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0149] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0150] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0151] Using such arrays, the present invention provides methods toidentify the expression of the enzyme proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the enzymegene of the present invention. FIG. 3 provides information on SNPs thathave been identified in a gene encoding the enzyme protein of thepresent invention. 41 SNP variants were found, including 7 indels(indicated by a “−”) and 4 SNPs in exons, of which 2 of these causechanges in the amino acid sequence (i.e., nonsynonymous SNPs). Thechanges in the amino acid sequence that these SNPs cause is indicated inFIG. 3 and can readily be determined using the universal genetic codeand the protein sequence provided in FIG. 2 as a reference SNPs,identified at different nucleotide positions in introns and regions 5′and 3′ of the ORF, may affect control/regulatory elements.

[0152] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol.2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0153] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0154] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0155] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0156] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified enzyme gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0157] Vectors/Host Cells

[0158] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0159] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0160] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0161] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0162] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0163] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0164] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0165] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0166] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0167] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0168] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0169] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enteroenzyme. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0170] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0171] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0172] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0173] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufinan etal., EMBO J. 6:187-195 (1987)).

[0174] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0175] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0176] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0177] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0178] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0179] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0180] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0181] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell- free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0182] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asenzymes, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0183] Where the peptide is not secreted into the medium, which istypically the case with enzymes, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0184] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0185] Uses of Vectors and Host Cells

[0186] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga enzyme protein or peptide that can be further purified to producedesired amounts of enzyme protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0187] Host cells are also useful for conducting cell-based assaysinvolving the enzyme protein or enzyme protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native enzyme protein is useful forassaying compounds that stimulate or inhibit enzyme protein function.

[0188] Host cells are also useful for identifying enzyme protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantenzyme protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native enzyme protein.

[0189] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a enzyme proteinand identifying and evaluating modulators of enzyme protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0190] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the enzyme proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0191] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the enzyme protein to particularcells.

[0192] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0193] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0194] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0195] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, enzyme protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo enzymeprotein function, including substrate interaction, the effect ofspecific mutant enzyme proteins on enzyme protein function and substrateinteraction, and the effect of chimeric enzyme proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more enzyme proteinfinctions.

[0196] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 1382 DNA Homo sapien 1 cctggaagtg ccagggagca ctggaggcca cccagtcatgggggacacct tcatccgtca 60 catcgccctg ctgggctttg agaagcgctt cgtacccagccagcactatg tgtacatgtt 120 cctggtgaaa tggcaggacc tgtcggagaa ggtggtctaccggcgcttca ccgagatcta 180 cgagttccat aaaaccttaa aagaaatgtt ccctattgaggcaggggcga tcaatccaga 240 gaacaggatc atcccccacc tcccagctcc caagtggtttgacgggcagc gggccgccga 300 gaaccgccag ggcacactta ccgagtactg cagcacgctcatgagcctgc ccaccaagat 360 ctcccgctgt ccccacctcc tcgacttctt caaggtgcgccctgatgacc tcaagctccc 420 cacggacaac cagacaaaaa agccagagac atacttgatgcccaaagatg gcaagagtac 480 cgcgacagac atcaccggcc ccatcatcct gcagacgtaccgcgccattg ccaactacga 540 gaagacctcg ggctccgaga tggctctgtc cacgggggacgtggtggagg tcgtagagaa 600 gagcgagagc ggttggtggt tctgtcagat gaaagcaaagcgaggctgga tcccagcgtc 660 cttcctcgag cccctggaca gtcctgacga gacggaagaccctgagccca actatgcagg 720 tgagccatac gtcgccatca aggcctacac tgctgtggagggggacgagg tgtccctgct 780 cgagggtgaa gctgttgagg tcattcacaa gctcctggacggctggaaag acgacgtcac 840 aggctacttc ccgtccatgt acctgcaaaa gtcagggcaagacgtgtccc aggcccaacg 900 ccagatcaag cggggggcgc cgccccgcag gtcgtccatccgcaacgcgc acagcatcca 960 ccagcggtcg cggaagcgcc tcagccagga cgcctatcgccgcaacagcg tccgttttct 1020 gcagcagcga cgccgccagg cgcggccggg accgcagagccccgggagcc cgctcgagga 1080 ggagcggcag acgcagcgct ctaaaccgca gccggcggtgcccccgcggc cgagcgccga 1140 cctcatcctg aaccgctgca gcgagagcac caagcggaagctggcgtctg ccgtctgagg 1200 ctggagcgca gtccccagct agcgtctcgg cccttgccgccccgtgcctg tatatacgtg 1260 ttctatagag cctggcgtct ggacgccgag ggcagccccgacccctgtcc agcgcggctc 1320 ccgccaccct caataaatgt tgcttggagt ggaaaaaaaaaaaaaaaaaa aaaaaaaaaa 1380 aa 1382 2 386 PRT Homo sapien 2 Met Gly AspThr Phe Ile Arg His Ile Ala Leu Leu Gly Phe Glu Lys 1 5 10 15 Arg PheVal Pro Ser Gln His Tyr Val Tyr Met Phe Leu Val Lys Trp 20 25 30 Gln AspLeu Ser Glu Lys Val Val Tyr Arg Arg Phe Thr Glu Ile Tyr 35 40 45 Glu PheHis Lys Thr Leu Lys Glu Met Phe Pro Ile Glu Ala Gly Ala 50 55 60 Ile AsnPro Glu Asn Arg Ile Ile Pro His Leu Pro Ala Pro Lys Trp 65 70 75 80 PheAsp Gly Gln Arg Ala Ala Glu Asn Arg Gln Gly Thr Leu Thr Glu 85 90 95 TyrCys Ser Thr Leu Met Ser Leu Pro Thr Lys Ile Ser Arg Cys Pro 100 105 110His Leu Leu Asp Phe Phe Lys Val Arg Pro Asp Asp Leu Lys Leu Pro 115 120125 Thr Asp Asn Gln Thr Lys Lys Pro Glu Thr Tyr Leu Met Pro Lys Asp 130135 140 Gly Lys Ser Thr Ala Thr Asp Ile Thr Gly Pro Ile Ile Leu Gln Thr145 150 155 160 Tyr Arg Ala Ile Ala Asn Tyr Glu Lys Thr Ser Gly Ser GluMet Ala 165 170 175 Leu Ser Thr Gly Asp Val Val Glu Val Val Glu Lys SerGlu Ser Gly 180 185 190 Trp Trp Phe Cys Gln Met Lys Ala Lys Arg Gly TrpIle Pro Ala Ser 195 200 205 Phe Leu Glu Pro Leu Asp Ser Pro Asp Glu ThrGlu Asp Pro Glu Pro 210 215 220 Asn Tyr Ala Gly Glu Pro Tyr Val Ala IleLys Ala Tyr Thr Ala Val 225 230 235 240 Glu Gly Asp Glu Val Ser Leu LeuGlu Gly Glu Ala Val Glu Val Ile 245 250 255 His Lys Leu Leu Asp Gly TrpLys Asp Asp Val Thr Gly Tyr Phe Pro 260 265 270 Ser Met Tyr Leu Gln LysSer Gly Gln Asp Val Ser Gln Ala Gln Arg 275 280 285 Gln Ile Lys Arg GlyAla Pro Pro Arg Arg Ser Ser Ile Arg Asn Ala 290 295 300 His Ser Ile HisGln Arg Ser Arg Lys Arg Leu Ser Gln Asp Ala Tyr 305 310 315 320 Arg ArgAsn Ser Val Arg Phe Leu Gln Gln Arg Arg Arg Gln Ala Arg 325 330 335 ProGly Pro Gln Ser Pro Gly Ser Pro Leu Glu Glu Glu Arg Gln Thr 340 345 350Gln Arg Ser Lys Pro Gln Pro Ala Val Pro Pro Arg Pro Ser Ala Asp 355 360365 Leu Ile Leu Asn Arg Cys Ser Glu Ser Thr Lys Arg Lys Leu Ala Ser 370375 380 Ala Val 385 3 18853 DNA Homo sapien misc_feature (1)...(18853) n= A,T,C or G 3 tactaaaaat acaaaattag ccaggcgtgg tggcgcacac ctgtaatcccagctacttgg 60 gaagctgagg caggagaatc gcttgaacct ggaaggcaga ggttgcagtgagccgagatt 120 gtgccactgc actccagcct gggcaacaag agcgaaactt cgcttcaaacaaataaatta 180 acgcccagca tgtcttggct ttcatctgcc agacctcaac cctcacccccaggagatcag 240 gtccggacca tgagctgacc ctggactcag gcaagggtga gttggtgcagccctggcctg 300 ctgggaggca caggctgcag caggctgcct ggggctgagg cccgccactcatgaactcat 360 gaccttgaat gagctccaaa agctctgggc ctcccaggct ctagggggagtgggagagag 420 aggcctcagc ctgtccctgg gcatgctgcc ccctcctcac ctctttgtcccaaatcccct 480 tcctggcaaa gctgacagtc ttaatatcac tctggagaaa actgagtcagccctaaggaa 540 caattcaatg aaccatttgc ttacttgagg attggaactc aagtctcactcaaagtctgt 600 gccattttcg tcccagctgt cactggccct catccacaca cacccaaggatgagcatcta 660 acgcttgcat gcacactccc atgcccgcgt tcattcactc attcattcattcattcactc 720 attcattgac tcattcattc attcactcac tcattcattc actcagtgaatgttgcagtc 780 acgatccaaa tatttatggc ctctgtgtgc caggcactag atggaggggctggggctaga 840 gcccctgata acccggtcat gccctagctt tcctgggaca cacattgtggtaaggggaga 900 ctaaaaaaat taagtcaggc caggcacggt ggctcatgcc tgaatcccagcactttggga 960 ggccgaggcg agtgaattac ctgaggtcag gagttcaaga ccagcctggccaacatggag 1020 aaacccagtc tctaattaaa aaaaaaaaaa aaattagcca ggtgtggtggcacatgcctg 1080 taatcccagc tactcaggag actaacgcaa gagaattgct tgaacccaggaggcagaggt 1140 tgcggtgagc cgagatcgcg ccattgcact ccagcctggg aaacaagagcgagactccat 1200 ctcaaaaaaa aaaaaagtgg gaggcagagg caggaggatc actagaggccagtagtttga 1260 gaccatcctg ggcaacatag caggaccctg tctgtacaaa aaaattaaaaaaaatttaac 1320 cgggcatggt ggcacacacc cgtagtccca gctactccag aggctgaggcaggaggatcg 1380 ctggagccca ggagttggag gctgcagtga actgtgatcc caccactgcacttaagcctg 1440 gataacaaag caagaccctg tctcaaataa caatagcaat aataataaagaaaaattaaa 1500 tgcaatttgc gatgcatcag tgataagtgc tctgcagaaa aaggaggcaggaagaggctg 1560 agaaaggtat gaggtttgct atgcaatgtg aagttatcaa ggaaggcttctcggaagagg 1620 tgacatttga gcagagaaat ggaggagagt tatggaggga agatggtgaatggggggaac 1680 atggtcaaga ccaggaatat ggtcaagggg ggaaagatgg tcaaggggacgcagcaaatg 1740 caaaggccct gaggcaggag cagcttgatt cacccccaaa acccgtggggcccgtgcagg 1800 cgacgggaag gacaagtgta aacccttttc cttgtccctg caggtgtgtgtgaacatgag 1860 tctgcccatg tttacaccct gcaagcctga agagtcccca gaaactgaaagaagaagcaa 1920 agccctttct gtaccctccc tgccccctgt cccgaccgcg acaaaagcgacttcctcttt 1980 ccagtgcatt taaggcgcag cctggaagtg ccagggagca ctggaggccacccagtcatg 2040 ggggacacct tcatccgtca catcgccctg ctgggctttg agaagcgcttcgtacccagc 2100 cagcactatg tgagtagctg gtggagggca tcccgtgggg ggaatacgggagggacagca 2160 cggccaccct tgcagtccca gggccaacca gctccagtga ggactaacggggcagggtct 2220 tgggcacctg gtccctggtc tttgagcctg gatctacccc tctgatccctgggaagacag 2280 ttcccttgga cccgccctgg gccccagccc tttactgtcc ccgcctgtgtccccagccag 2340 gccctcagcc ttagccagga gtcctctttc tgctcccctg ccatggccaggcagcccagc 2400 gctctctcag gtccgaggcc cactcctcca ggaagccttc cctgactagcccagctatca 2460 gagagtggcc ctcccaagag ggaggcctgg aaactaaagc tctctctctccccagctgcc 2520 tgtagtgtca gttagagtct tatcctctcc agtagggtga caccatgacaggggccaata 2580 gagtcctccc atctgtcccc aaggaggctg gacaaatgcc tgctcagacacacaagtcca 2640 ctgggtcccc taatcccata ggaaggccag ggaggaacta catttaggaaattgaagctt 2700 gtatggaaca tttagtccta tgtgccaaga cctttctctt ttttgttatttttttgtgtt 2760 ttgagacaga gtcttgatct gttgcccagg ccagagtgca gtggcacgatctcagctcac 2820 tgcaacctcc gccttccagg ttcaactggt tctcctgcct cagcctccagagtagttggg 2880 attacaggtg cccaccacca cgcctggcta atttttgtat ttttagtagagacagggttt 2940 caccatgttg gccagactgg tctcaaactc ctgacctcaa gtgatccacccacctgggcc 3000 tcccaaagtg ctgggattac aggcatgagc caccgtgcct ggcctgtttttttgaaatga 3060 ggtctggagt gcagtggtgc gatcatagtt cactgcagcc tcaacctcccaggcccaagt 3120 gatcctcctg cctcagcccc ttgagtagct ggggctacag gcgcacaccaccatgcctgg 3180 ctagttttta aaatttttgt ggagatgagg tttcactatg ttgtccaggctaatcttgaa 3240 ctcctcggct taagcaaccc tctggtctca gcctcccaca gtgctaggattacaagcgtg 3300 agctaccgtg cctagtcact tttctccttt tctttgtaac tttcagttttgaaatttcaa 3360 atttacagaa aggctactgg gtgtcaaaac ggtaccagtc actccaatagtctttcactc 3420 accttcatcc acacctctct ttctggggat attttctgaa ttatttgagagtgagttgaa 3480 gacgtgtttc tttacctcta aatactagtt gttgggcatt tcttaaaatcaaggcattct 3540 cttacataat cacaacacac gtgtcaaaat caggaaatta acatggacaaaacaccatta 3600 tccacccaca gactttactg aggtttcccc gattatcctg cttgtcctctgcagtgaaaa 3660 cttttttcag gtctaggatc cagtcaagga tcaatgtcat agcctttaaccttctttaat 3720 ctggatcagt cttttttctt tttctttttc tttttttgga cacggaatctcactctgtcg 3780 ccagactgga gtgcagtggt gcaatctcgg ctcattgcaa cctctgcctcctgggttcaa 3840 gagattctcc tgcctcagcc tcctgagtag ctgggaatac aggtgcgcgccaccacgccc 3900 agctcgtttt tggtagagac agggttttgc cattgattct ggatcagtcttttttttttt 3960 ttttatgaga tggagtctta ctctgtcacc caggctggag tgcaatggcacaatctccac 4020 tcactgcatc ctccgcctcc caggttcaag caattctcgt gcctcagcctcccgagtagc 4080 tgggattaca ggcatgcgcc accatgcccg gctacttttt gtatttttagtagagacagg 4140 gtttcaccat gttagccagg ctgatctcga actcctgacg tcaggtgatctgcccgcctc 4200 gacctcccaa agtgctggga ttacaggcgt gagccaccgt gccagcggattctggatcgg 4260 tcttaatcag tctttgtctt ttgcaacttt gatgttttgc agagagcagaccagttacct 4320 tgtagaatgt cccttagttt gggtttatct tcattagatt cagtttgtgtatccagggca 4380 gtggatctta gatgcaattc tgtcttcttt ttaatttttt tgagagggagtctcgctctg 4440 tcacccaggc tggagtgcag tggcacaacc tcagctcact gcagcctccgcctcccgggt 4500 tcaagcaatt ctcctgtccc agcctcccaa gtagctggga tcacaggtgcccatcaccac 4560 taccgggtaa tttttgtgtt tttagtagag acagggtttc accatattggtcaggctggt 4620 cttgaacgcc tgacctcagg tgatccacct gccttggcct cccaaagtgctgggattaca 4680 gacgggagcc aacatgccca gccttcctgc ccctcccgtc ccctcccctctcctcctgtc 4740 ccctcccttc ccctccccta tcctcatgtc ccctcccttc ccctcccctccccacccaag 4800 ctggagtgca gtggtgcaat catagctcac taaagccttg acctccaagtctcaagcaat 4860 tctcctgcct cacctggggc cacaggtgtg cggcaccaca cccggacaatttttgtgttt 4920 ttagtagata tgggggtctc gctatgttgc ccaggctggt ctcaaactcttggactcaag 4980 cgatcttccc acctcggtac taaaaagtgc tgggattcca ggtgtgagccaccgtgccca 5040 gcctaggtcc tacttttatc tccaatttac agatgagtcc atttgagagaagctgaccct 5100 cttgccctgg gtctcaaggc tggggcgtgg cagcacttgg gtccacgtttgtgccctttc 5160 tgcaatccag gacaactgca aagatggtcc tcaccccaat cctctgggcttcctccagtg 5220 ggtagtggga tcctgggtgc acacagcaaa gcctctttgg aggctgaatggggtcccccg 5280 actctggctt tcccccaggt acatgttcct ggtgaaatgg caggacctgtcggagaaggt 5340 ggtctaccgg cgcttcaccg agatctacga gttccatgtg agtgtggggacggaggaggg 5400 acagggaccc accgttccag ctccaccctt tgggaaggac cttagcccaggtgatgggga 5460 aactgcagaa cccagaatcc cctcccagac cacagttaaa ggggatttatttatttatat 5520 aaatttttgt gacagggtct tgctctgtca cccagggtct tgctctgtcaccactctgaa 5580 cacctcatgt tctctgatta caggcatgag cccccacggt cggccttttaggtggttttg 5640 agaggtattt aggtttgcag tgcaggggcg caatcatagc tcactgcagcctcaacctct 5700 ggggctcaag cgatcctcct gcctcagcct cctgagtagc tgggactataggtgcgcatc 5760 accatgtgtg gctaattttt gtatttttta taaagatggg gatctcactatgttgcccag 5820 gctggtcttg aactccagac ctcaagtgat cctcctgcct tggcctcccaaagctaaggg 5880 ggcattaaaa gaaaaaaaca tttttccccc tgaaacattt aagtagtcttactgaaaaca 5940 ataaaacaca gaaacaccag attctcattt taaagtaaaa cagacaggatctcccagaac 6000 cttcctagaa tggaaccatt cttgtcgctt ttgaaaaaca aagccaagttctagatccca 6060 aataaatgca cctgctggtg aacattctcc ttgtggttct cgtccctatgttagttattt 6120 tcctaaattt tacatttgta cctttttaag aatgagttat cagtttttttatatttgctt 6180 ttcttttgag atggggtctt gctctgtcac ccaggctggg gtgcagtggtgcaatcacgg 6240 ctcactgcag cctcaacctc cagggctgaa gcgattctcc catctcagcctcccatgttg 6300 agatcacagg tgtgcaccac cacacctggc tccttttcct gatttgttttttgtagagat 6360 gggatttcgc tatgttgccc aggctggtct ctaactcctg gactcaagtgatcctcccgc 6420 ctcagcttcc caaattgcta ggattacagg tttgagcccc tgcacctggtcaacctgagt 6480 tttaagagga tccctttggc gactggattg aggacagaca agagtggacgggggacacaa 6540 ggaggccatt ttcgttatcc aggcctggta gtggctaggg ccaggagggtggggttggtg 6600 ggaagcagtc agatcccaaa gagatttggg gattggaagc aaaaggatttgctggtgact 6660 tgcacatggg agggagagag gtcagtgcct ctgttaatca aggaatccagattgccaccg 6720 aaatttctag gcccgagata tttaggtagt gtctcactct gtcacccaggatggagtgca 6780 gtggcgccat ctcggctcac tgtaacctcc gcctcccagg tttaaacgattctcccacct 6840 cagcctcctg agtagctggg attacaggca tgtgccacca ctcccggctaatttttgtat 6900 ttttagtaga gacggggttt caccacgttg gccaggctgg tcttgaactcctgacctcaa 6960 gtgatccacc cacgacagcc tcccaaagtg ctgggattac aggcgtgagccaccatgctc 7020 ggccttttag gtggttttga gaggtattta ggtcacttcc aatctcgtgcttttccaagt 7080 gttgtaaact acaaatattc cttcacgtct tcttgtcttt ttaatgtttagaaaacctta 7140 aaagaaatgt tccctattga ggcaggggcg atcaatccag agaacaggatcatcccccac 7200 ctcccaggtg agcacggggc tgagccgcct gtcagggggt cattggcgggggctcacctg 7260 ccctcccagc acctctcggg cttgacctca tgttctctgg tgccagctcccaagtggttt 7320 gacgggcagc gggccgccga gaaccaccag ggcacactta ccgagtactgcagcacgctc 7380 atgagcctgc ccaccaagat ctcccgctgt ccccacctcc ttgacttcttcaaggtgcgc 7440 cctgatgacc tcaagctccc cacggacaac cagtgagtga acttttcaccctgccaggtg 7500 ggagagggaa ggaggggtgg gactttctgt gttttgcaga tgaggaaaccaaggctcaga 7560 gagggaaagc caccttccca gagccacaca gccagaaaga ggaggcaaattccacctccg 7620 gcccctgtga ccccgccaag cctccacctt aatctttcac acctcagggcactgggggaa 7680 gcactcgggg ctggaggttc aaagtcctgg gtcctcatcc tgacattatggccacctggc 7740 catgggacct ggagccagtc accactgctc tctgaatgca ggttctccatttctataatg 7800 ggcagtgagg atcagatgaa gcattgggtg tcttgcggag ccccccagaaggatgtgggg 7860 ttgatgcctc tgctaagtgc tgagcatgtc tggggtctcc tgtacccaggaccctgtgtg 7920 gaaggcacct gagaggctga gggagctcca ggcaggctgg ggaagtccccttctccactc 7980 ctctctggtc actgaagctc gaagtgggga gcatgaggac aggacgttaccccttgtcaa 8040 ggcacccagg ctgccaagac agagacaagc agcattgctc cggccagcacttattgacgc 8100 ttgaaggtgt cccctggccc aaggaagggc agttatcatc agcccgggaggcgggggaag 8160 gatggactct gcagtggggt ccgctcctca ttgcctgctc tctcagggctccagaaggag 8220 gaagaggccg ggcacagtgg ctcacaccta taatcccagc actttggaaggtcgaggtgg 8280 gcagatcacc tgaggttggg agtttgagac cagcctggcc aacatggtgaaaccccatct 8340 ctaccaaaaa tataaaaatt tagtcaggca tggtggtgtg cgcttgtaatcccagctact 8400 tgggaggccg aggcaggaga atcgcttgaa cccgggaggc agaggttgcagtgagctgag 8460 actgcgccac tgcactccag cctgggtgac agagcgagac tctgtctaagaaaaaaaaaa 8520 gaaaagaaga aagaagatgg cctgggagcc cgcaagagca ttttccaggcttagggcatc 8580 ctttgggtct gcagaaggct atgcagtgtc ctcctcatgt ccctcccttgggctgcccga 8640 gcagatccgc ccgcccccat cacttcctga agcccttcct cagccagtccagttgctgtc 8700 ttctctccgc agtgcccctt ccctttcccg ggtccctctt ctcttgggaagttcttctgc 8760 aggtctaccc agtgcctctt cttcctccat gggaagccaa gggtctcacccagactgttc 8820 tctcctcagg acaaaaaagc cagagacata cttgatgccc aaagatggcaagagtaccgc 8880 gacaggtgag aggacggggg gcagccggcg gggggggaca ccctgaggagacccagagtg 8940 ttcagggaat ggagcagggg ctgggagcag gctgggaggg ctcacagctaccctgctgaa 9000 gaattgggtc tttgggccgg gtgcggttgc tcatgcctgt aatcccagcagtttgggagg 9060 ccgaggcagg tggatcactt gaggtcagga gtttgagacc agcctggccaacatggagaa 9120 accctgtctc tactaaaaat ccaaattagc caggcgtggt gacaggtgcctgtagtccca 9180 gccacttggg aggctgaggc aggagaattg cttgaacccg gaagacggagtttgcagtga 9240 gccgagatcg tgccactgca ctccagcctg ggcagcagag ccagactccatctcaaaaaa 9300 aaaaaaaaaa aagaagaatt gggtctttgg aaggtccctg gagactgaaaggagcccttt 9360 gcaggtggca gtgcagagac cagcgcagac ccttgctact ggcagccgggggagtgtttg 9420 cggctgaatg aatgaacagg ttttggaggg cagcgtggcc ttcagaggcgatgcagggct 9480 gtggcagttt ctaatactta ttgcacagtc actgctaata acaataataataataatacc 9540 taacattaat ggagtgctta ctctgtgcca gccactattt tgtttttgttgttttcagtg 9600 acagggtctc gctctgttgc ccaggccaga gtgaagtggt gtgatcatagctcactacag 9660 cctcgacctc ctgggctgaa gcgatcctcc cacctcagcc tcccaagtagctgggattac 9720 aggtgtgtgc caccatgtcc agctaatttt taattttctg atagagatggggtctcacta 9780 cattgcccag gctggtctta agctcttggc ctcaagcaac cctcctgcctcagcctccca 9840 aagtgctgag attatagaca tgagccactg tgcccggctt tttcttcttcttataaggac 9900 acgaggcctg ttgggttagg gcccactcta ctgacctcat tttaacttaattacctcttg 9960 aaacgtactt aagagtacct ttctcttaat acacccacac tgtaaggtactgggtggtta 10020 ggacttcaac atatgaattt tgagaaggcg gatgtcagcc aataccaaacagcatcagca 10080 cctccacggt tggatgaagg gctggtcaga aatgcacact caggtcccacagtggaccta 10140 ctgaacagga taggcatttt agcaaaatcc caggtattcg ggtgcaccttaaagttagga 10200 aaaggtcagg cactgtggct catgcctgta atcccagcac tttgggaggccgaggcggtt 10260 gaatcacctg aggtcaggag ttcgagacca gcctgaccaa tatcgtgaaactccatctct 10320 actaaaaata caaaaattag ccaggtgtgg tggcgggtgc ttgtagtcccagctacttgg 10380 gaggctgagg caggtgaatt acttgaacct gggaggtgga ggttgcaatgagccaagatt 10440 gcaccactgc actccagtga cagagcgaga ctccatctca aaaaaaaaaaaaaaaaaagt 10500 tgggaaaagg ccaggtgcag tggctccacg cctgtaatcc caacactttaagaggctgag 10560 gtgggagaat cctttgagcc caggagttcg agaccagcct gggcattgtcccaagacctt 10620 gtctttacaa aaaattagcc gggtgtggtg gcatacgtct gtggtcccagctattcggga 10680 ggctgaggca gggagattgc ttgagcctag gagtctaggg ctgtagtgagctgtgatcac 10740 gtcactgtac tctagcctgg gcaacagagc aagactctgt ctccaaaaaagaaaataaag 10800 ttgggaaagg ctcactaact tcatcagatg agaacaaaga catgtttgaagtgtgaggcc 10860 gaagcctgga gaacgctatg cgcccaggaa atgcagggca gcagagactcaagatgccag 10920 cgcctgttct ggaggcccag atgggccctg caatgcccac tcaccctgccctccctcttg 10980 ccccagacat caccggcccc atcatcctgc agacgtaccg cgccattgccgactacgaga 11040 agacctcggg ctccgagatg gctctgtcca cgggggacgt ggtggaggtcgtggagaaga 11100 gcgagagcgg tcagacctcc caccttacgg ggctccttcc cctggtgctcaggaacccac 11160 agccacaagc cccctgccaa ggctcaggca gcctggcccc tgggaggactccagctctgt 11220 taggggccct aaatgtcctc cccacactgt gggtcgcctt ctctcttagtgtgcaccctg 11280 tggtggctgt gggcatctgt gcatggcagg ccggggcggg gcatgtctgcgtgttctgtc 11340 tggatgggta tgggaccgtc tgttcattat gaagtgggct cagagctgtgattctgtgag 11400 catgtgtgca tgcatgcatg tgacctcatt gtccagtgtg gtgaaggtgacatttccaaa 11460 tctgagcatt ggacatcagt gtgtctgtgt ccctgtgtcc tcaccatccctgatggctgc 11520 agggagccgc tgggccctgc ccctcagtca cattcccgca cctctggcacaggttggtgg 11580 ttctgtcaga tgaaagcaaa gcgaggctgg atcccagcat ccttcctcgagcccctggac 11640 agtcctgacg agacggaaga ccctgagccc aactatgcag gtgccccctgccctccgagg 11700 ctgtaggggt gtgggagaaa ggggcaggca gggctcaggg atattgagtgactgctttgg 11760 agtctgggct ggttgctggc ttggcagaaa agtcagggct aagatctcatcggctctggc 11820 ttgggggccc tggcaggttg tgatgccctt ggtctggaca gggaaccaggaggaggagca 11880 gacgactcgg gagagtggga ggccagtggt gtctgtggat atgtggccaggttcagtggg 11940 aagctgaagg atgagcagac cttaggctca ggaaggaggg ctgcctggaagtgggggcat 12000 catcactgac cagaaaggga aaactggcag tgccagggct ggatggggcctgcattgagc 12060 ttgaaaaaaa ctataataga attggttacc attttatttt attatttatttatttatttt 12120 acttttttga gatagagtct cactcccttg ctaaggctgg agtgcggtggtgctatctca 12180 gctcactgca acctctgcct cccaggatca agtgattctc cagcctcagcctccccaggt 12240 agctgggatt acaagcatgc accaccatgc ctggataatt tttgtatttttagttgagac 12300 ggggtttcac caggttggcc agactggtct cgaacttctg acctcaggtgatctgcctgc 12360 ctcggcctcc caaagtgctg gaattacaga tgtgagccac tgtccctggcctggttaccc 12420 acattttaaa atggagtgat ttcacccttt tatgtggatt tacagcttgttttttttttt 12480 tttttgagac aaagtctggc tctgtcaccc aggctggagt gcagtaatgcaatctcagct 12540 cactgcaacc ttagcctcct gggttcaagc aattctcctg cctcagccacctgagtagcc 12600 tggggttaca ggcatgcacc accacgccag gctaattttt tgtatttttagtagagatgg 12660 ggtttcgcca tgttggccag gctggtctcg aactcctgac ctcaggtgatccgcccgcct 12720 tggcctccca aagtgctagg attacaggtg ggaaccacct tgcccagcctgtggctatcg 12780 tttaaacact gggaaggcct gcagccccca ggccgacagt tagctgcagctgagcagttc 12840 ccagtgccag gtagacggat gctccaccca cctactcatg gctgatctcttgtcatagtg 12900 aagtgtctgg acagaccttc atcgttatgg gatctctggt ccccagagtgggtggcaatg 12960 aatgggagtg gacaagctca cctgggtgta gggggcagag ggccgaagtccagagtgtac 13020 ccccagagtg ggtgccagca ggagcttgcc gagggatctg ggatggagcaggagggtgga 13080 gggaggagac ccagaagagg gggaactgtg ggccctgggt gggtctggagtgcctggagg 13140 aagcccaggc gcagagagga gaagatggga tgggtggcga gccccaggctgggccgacct 13200 cacactgtgc tctgtgcccc tgccgtggac caggtgagcc atacgtcgccatcaaggcct 13260 acactgctgt ggagggggac gaggtgtccc tgctcgaggg tgaagctgttgaggtaattc 13320 acaagctcct ggacggctgg tgggtcatca ggtaggaggg cccctctccatccagagcac 13380 ccatctgagt cagccccagc caggacgggg tgtttaggga tctggggtgacttgtccctg 13440 ggactctggg taagccactg cccctctctg ggcttagttt ccatctcagtagcagggagg 13500 gatgagccca cccttgcctg tcttgtgggg atccaatgtc cttgtccaagtgggtgcatt 13560 tctcctttgt gatttagggt ctcttcccaa ccatctatta ttattccttctctggcaaca 13620 tggtgaactg ttgtataaat aattacattc ctagctaggc gcaatggcccaggcctgtaa 13680 tcccagcact ttgggagccc aggacaggac gatcacgtga ggtcaggagttcgagaccac 13740 cctggccaac atggcaaaac cctatctcta ctaaaaacac aaacatgagccgggtgttgt 13800 ggtgggagcc tgtaatccca gctactcggg agtctgagac aagagaatcacttcaacccg 13860 ggaggcggag gttgcagtga gccaagatcg cgccattgca ctccagcctgggcaacgaga 13920 gcgaaactcc gtctcaaaaa aaaaaaaaaa aaaaaagatt actttctttttatcattcct 13980 ttatctttta aagctttctt gcagtcaggt gcagtgtctc atgcctgtaatcccaacact 14040 ttgggaagct gaggtgggag gatcactcaa ggctacaagt tcaagaccaacctgggcaat 14100 gtagggagac ctctgtctct acaaaaaaaa ttaaaaaata gctggatgtggtagcacaca 14160 cctgtagccc cagctactca ggaggctgag gtgaaaggat cacttgaccccaggagttgg 14220 aggcagcagt gagctatgac tgcaccactg caccccagcc tgggtgatggagcaagaccc 14280 tgtctcaaaa aaaaaaaaaa aaaaaaagct tccattgcaa ttcccatctgtttatcctcc 14340 aaatgaatgc agaaatacta attatctttt ttctggttct ggggaacacagaattctagc 14400 ggcttgtgga gccatttccc tggagccatg gggcctccca ggtcctttcctgtgtcttca 14460 ttttttacga attttttcat tttttgagac aggatcttgc tctgactcccaggctggagc 14520 acaatcatcg ctcactcaag cgatcctccc acctcaggct cccacgtagctgggactaca 14580 ggtgagcacc accacatctg gctaatgttt tttaattttt ttgtaggggtggggtctcac 14640 tatggtgcca agactagtct taaactcctg gcctcaagag ttcctcctgccttggcctcc 14700 caaagcactg ggattacagg aatgagcctc catgctgggc ctttgctggcgtcttcagag 14760 ccctaggtca cagggccagc ctggcgccct gccgcaagct tatcttaaagctgggaccac 14820 aacatgcata cctgcagccg ggcccggggc cagagggctt tgaggcagcatttctcagcc 14880 ttttagacac acactctgtt aacccccatc ctgtgtctct gataatcttcttgtgatcct 14940 cccaccagcc aagaattggg ttttatgtga accttgtatt atgcaaagttttcttttgtt 15000 ttttttttca ctcccaaata taatattgag aatagaaaga aagtcttttcaacaaatggt 15060 gctggaacag atggatttcc atactggaaa aaaaaaaaaa agagcaaaaaacaaacctag 15120 accccttcct cacactgtac acatatgttt acttcagatg gatcacaggtttatcccaga 15180 gtaaaacctg aaactaaaaa ccatttgggg ctggacaggg agctcacgcctgtaatctca 15240 gcactttggg aggctgaggc aggtggatca cttgatgtca ggagtttgagaccagccatg 15300 accaatatgg tgaaatcctg tctctactaa aaaaatacaa aattaaccaagtgtggtggt 15360 gcatgcctgt aatcccagct acttgggaag ctgagacagg agaattgcttgaacttggga 15420 agcagaggtt gcaatgagtc gacatcatgc cattgcactc cagcctaggcaacaagagca 15480 aaactctgtc ttggggttgg gtgggggaaa agcatttgga agaaagcatagaatttggtg 15540 gcttggaggt aggcaaaggt tcgtaggaga cagaaggcag ttaacataaaagaaaaattg 15600 gcaaatataa tcctgccagt gtcttctttt ttctttaatt ttttcgggaggtagagatag 15660 gggtcttgct atgttaccca ggctgatctc caactcctgg cctcaagcgatcctcccacc 15720 tagatccctc aaagtactgg gattacaggc gtgagcgacc gtgccctgcccattcttgcc 15780 aatgtcttat agcaaatacc tgtcccctgc ggtgacctgg atctgctaacctccacccct 15840 gcctagactg tggaaggatt gctggaaggg tctcagttgc acagaccaggaaactgaggc 15900 ccacagaggc aggtgtccgg ttgtttgcaa cctctcagcc tgtgctaaccccaattgttc 15960 agagagagcc ctgaaaccct ctcctctggg cgcccccagg tgactgccccagcctcaagg 16020 gctgcctctg ttgcaggaaa gacgacgtca caggctactt cccgtccatgtacctgcaaa 16080 agtcagggca agacgtgtcc caggcccaac gccagatcaa gcggggggcgccgccccgca 16140 ggtaagcggg ggtccccggg gctgggcggg gtcgagcggg gcgcaccacgggttcgctct 16200 gtctaggcca tagcttggca gtgccggggc gggggctctc agcctggcaggagaggcagg 16260 accctcacgg gggaaagggg ctggacgcgc ctggccgcgg tgtggggctggcacgggggc 16320 ggaaggaaag cggcgatgcc cgggggcttt ggggatgggc agtccaggggggctccccgg 16380 agagggggac gacagaccga aggctggtga ggggcgtgga aaaccgcccaggctctgctg 16440 cagggcaagg gtccttgtcg tgacgggggc agccgcctct tgtcccgccggggtcgtgca 16500 gactaccggc cccctactgc cccccacttc ctcggaccag gggtgcccatctgagtccct 16560 gggggcaggg gcgccctcgg gctttgacga cgccccgtcc cgctgggccaggtcgtccat 16620 ccgcaacgcg cacagcatcc accagcggtc gcggaagcgc ctcagccaggacgcctatcg 16680 ccgcaacagc gtccgttttc tgcagcagcg acgccgccag gcgcggccgggaccgcagag 16740 ccccgggagc ccgctcggtg agtgcagcgg gagagggcag gaagggcaagccctaggggc 16800 ggagtcagcg ggagaggcgg ggccagaggc agggccagag tagcggggcgggaccagagg 16860 gcggaatcag agggagaggc ggggactgga ggcgggggca gaggaggagccagcgctagg 16920 gggcggagcg atccctaaga ggcggagtca gagggagagg cacaagcgggaggcgaggcc 16980 agagcgcgga gcaggagttg gagaccgcgg cggggcgagg ccagagagcgctgtgggcgg 17040 ggccagtgtg cggggcgggg cgtctgactc ggccccgctc tctgcccgcagaggaggagc 17100 ggcagacgca gcgctctaaa ccgcagccgg cggtgccccc gcggccgagcgccgacctca 17160 tcctgaaccg ctgcagcgag agcaccaagc ggaagctggc gtctgccgtctgaggctgga 17220 gcgcagtccc cagctagcgt ctcggccctt gccgccccgt gcctgtatatacgtgttcta 17280 tagagcctgg cgtctggacg ccgagggcag ccccgacccc tgtccagcgcggctcccgcc 17340 accctcaata aatgttgctt ggagtggacc gaggctctgc aggaatgcagggagggccgg 17400 gctccgcccc agggttattt tctaagttga ggacagggag gttgtgagttctgnnnnnnn 17460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 17940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 18000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 18060 nnnnnntaaa aattagctgg gcgtggtggc atgcatccac aatcccagctactggggagg 18120 ctgaggcatg agaatcgctt gaaccgggga ggcagatgtt gcagtgagccgagacggcgc 18180 cactgcactc cagcctggac tacagagcga gactctatct caaaaaaaaaaaaaaaaaaa 18240 aagtaactta ggtgcagggt gtcctctgtt attcactgag accgtgccccggttatgagg 18300 ttgtaccaga aagcaagtat tcactatgca cactattcac cgctcaccctagcattgaag 18360 ccagcctgta gcctgaaagc ctttgctttg agggcaggtc tttccccaaaatgcagacac 18420 gaaggtgcaa agtgaagctg ccagtcttgc aaaagatgta acttgtcacgaaggccacga 18480 gtggcaggga gagctgtccc acatttgcgg aagtggctat gtgaggacgggggaggcggg 18540 tcccttagag ataagagaca atcataaggg gagatatcag agaaaatcgtaaggggagca 18600 gatggttgtc aagagaatag gctgaccatc gaaggactgg cagaagctttcagaaaacca 18660 ctggacggct gggcacagtg gcttaggcct gtaatcccag cactttgggaggctgacgca 18720 ggtgaatcac ttgaggtcag gagttccaga ccagcctggc caacatggtgaaaccccatc 18780 tctacagaaa atataaaaat tagccaggcg tggtggcaca agcctagaatcccagctact 18840 tgggaggctg agg 18853 4 390 PRT Homo sapien 4 Met GlyAsp Thr Phe Ile Arg His Ile Ala Leu Leu Gly Phe Glu Lys 1 5 10 15 ArgPhe Val Pro Ser Gln His Tyr Val Tyr Met Phe Leu Val Lys Trp 20 25 30 GlnAsp Leu Ser Glu Lys Val Val Tyr Arg Arg Phe Thr Glu Ile Tyr 35 40 45 GluPhe His Lys Thr Leu Lys Glu Met Phe Pro Ile Glu Ala Gly Ala 50 55 60 IleAsn Pro Glu Asn Arg Ile Ile Pro His Leu Pro Ala Pro Lys Trp 65 70 75 80Phe Asp Gly Gln Arg Ala Ala Glu Asn Arg Gln Gly Thr Leu Thr Glu 85 90 95Tyr Cys Ser Thr Leu Met Ser Leu Pro Thr Lys Ile Ser Arg Cys Pro 100 105110 His Leu Leu Asp Phe Phe Lys Val Arg Pro Asp Asp Leu Lys Leu Pro 115120 125 Thr Asp Asn Gln Thr Lys Lys Pro Glu Thr Tyr Leu Met Pro Lys Asp130 135 140 Gly Lys Ser Thr Ala Thr Asp Ile Thr Gly Pro Ile Ile Leu GlnThr 145 150 155 160 Tyr Arg Ala Ile Ala Asp Tyr Glu Lys Thr Ser Gly SerGlu Met Ala 165 170 175 Leu Ser Thr Gly Asp Val Val Glu Val Val Glu LysSer Glu Ser Gly 180 185 190 Trp Trp Phe Cys Gln Met Lys Ala Lys Arg GlyTrp Ile Pro Ala Ser 195 200 205 Phe Leu Glu Pro Leu Asp Ser Pro Asp GluThr Glu Asp Pro Glu Pro 210 215 220 Asn Tyr Ala Gly Glu Pro Tyr Val AlaIle Lys Ala Tyr Thr Ala Val 225 230 235 240 Glu Gly Asp Glu Val Ser LeuLeu Glu Gly Glu Ala Val Glu Val Ile 245 250 255 His Lys Leu Leu Asp GlyTrp Trp Val Ile Arg Lys Asp Asp Val Thr 260 265 270 Gly Tyr Phe Pro SerMet Tyr Leu Gln Lys Ser Gly Gln Asp Val Ser 275 280 285 Gln Ala Gln ArgGln Ile Lys Arg Gly Ala Pro Pro Arg Arg Ser Ser 290 295 300 Ile Arg AsnAla His Ser Ile His Gln Arg Ser Arg Lys Arg Leu Ser 305 310 315 320 GlnAsp Ala Tyr Arg Arg Asn Ser Val Arg Phe Leu Gln Gln Arg Arg 325 330 335Arg Gln Ala Arg Pro Gly Pro Gln Ser Pro Gly Ser Pro Leu Glu Glu 340 345350 Glu Arg Gln Thr Gln Arg Ser Lys Pro Gln Pro Ala Val Pro Pro Arg 355360 365 Pro Ser Ala Asp Leu Ile Leu Asn Arg Cys Ser Glu Ser Thr Lys Arg370 375 380 Lys Leu Ala Ser Ala Val 385 390

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NQ:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human enzyme protein, said method comprisingadministering to a patient a pharmaceutically effective amount of anagent identified by the method of claim
 16. 19. A method for identifyinga modulator of the expression of a peptide of claim 2, said methodcomprising contacting a cell expressing said peptide with an agent, anddetermining if said agent has modulated the expression of said peptide.20. An isolated human enzyme peptide having an amino acid sequence thatshares at least 70% homology with an amino acid sequence shown in SEQ IDNO:2.
 21. A peptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human enzyme peptide, saidnucleic acid molecule sharing at least 80 percent homology with anucleic acid molecule shown in SEQ ID NOS:1 or
 3. 23. A nucleic acidmolecule according to claim 22 that shares at least 90 percent homologywith a nucleic acid molecule shown in SEQ ID NOS:1 or 3.